Portable device and method for enriching tumor exosomes

Abstract

The invention belongs to the technical field of exosome enrichment detection, and relates to a portable device and method for enriching tumor exosomes. The method comprises the following steps: mixing exosome serum with PBS (Phosphate Buffer Solution) and E-Am molecules to obtain a mixed solution, introducing high-pressure bubbles from the bottom of the mixed solution, floating out of the liquid surface to form foams, continuously introducing the high-pressure bubbles, rising the foams, collecting a stable foam group formed after the foams are lifted to a certain height, completing exosome enrichment, and collecting and storing the enriched exosome for later use or detecting and using. The invention provides a novel method for selectively enriching exosomes by taking bubbles as a carrier. Zero-damage, ultra-fast and economical sorting is realized, and the damage of a strong physical field to the exosome is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome enrichment detection, and relates to a portable device and method for enriching tumor exosomes. Background Art

[0002] Early diagnosis and treatment of cancer is the key to improving patient survival and prognosis. Taking lung cancer as an example, the five-year survival rate of advanced lung cancer is only 5.8%, while the five-year survival rate of early lung cancer can reach 92%. The development of early cancer diagnosis technology has important clinical value. Exosomes are a type of extracellular vesicles secreted by cells, with a diameter of 30-150mn, carrying a large number of nucleic acids and proteins related to mother cells. The heterogeneity of exosomes in cancer patients will increase, bringing new hope for the early detection of tumors. However, exosomes have small particle size, low buoyancy density, and low concentration of cancerous exosomes in the early stage of onset. How to efficiently enrich and accurately detect these exosomes is an urgent problem to be solved. Traditional exosome extraction methods such as ultracentrifugation and filtration methods have problems such as long time consumption, co-precipitation contamination, exosome rupture, and large sample loss, and it is impossible to achieve exosome extraction-detection integration. As for the exosome sorting technology based on microfluidics, as the particle size decreases, the control of microfluidics over exosomes rapidly decays, and it is necessary to increase the intensity of the physical field (electric field, acoustic field, etc.). Strong physical fields and long exposure time in the physical field will inevitably damage the exosomes, and the final sorting effect is not ideal, the sorting efficiency is low, and the product purity is low. Microfluidics technology based on immunoaffinity and magnetic nanoparticle separation technology are currently effective methods for obtaining high-purity exosome products, but exosomes captured based on immunoaffinity are difficult to elute and cannot meet the needs of downstream analysis. Therefore, it is urgent to develop new technologies that can efficiently enrich exosomes, improve detection automation, and avoid false positives caused by manual operation errors. Summary of the invention

[0003] Aiming at the problems existing in traditional exosome enrichment and detection, the present invention proposes a novel portable device and method for enriching tumor exosomes.

[0004] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions: Application of E-Am molecules in exosome enrichment and / or detection, wherein the molecular formula of the E-Am molecules is R1-R2-R3-COOH; wherein, R1 is a C15 straight chain alkyl group: CH3(CH2) 14 -; R2 is (CH2CH2O)4, and the structural formula is ; R3 is the following peptide chain: -Cys-Arg-His-Ser-Gln-Met-Thr-Val-Thr-Ser-Arg-Leu-, Among them, Cys is cysteine, Arg is arginine, His is histidine, Ser is serine, Gln is glutamine, Met is methionine, Thr is threonine, Val is valine, and Leu is leucine.

[0005] The application method is: exosome serum is mixed with PBS and E-Am molecules to obtain a mixed solution, high-pressure bubbles are introduced from the bottom of the mixed solution and then float to the surface to form foam, high-pressure bubbles are continuously introduced, the foam rises, and the stable foam group formed after rising to a certain height is collected to complete the enrichment of exosomes. The enriched exosomes are collected and stored for later use or detection.

[0006] Preferably, the volume ratio of exosome serum: PBS: E-Am molecules is (50-200): (1000-50000): 1.

[0007] The present invention also provides a device capable of realizing the above-mentioned application, comprising a bubble generator, a gas inlet being arranged on the side of the bubble generator, a cylindrical sample storage area and a foam purification area being arranged in sequence above the bubble generator, a sample outlet being arranged on the side of the top of the foam purification area, and a sample addition port being arranged on the upper part of the foam purification area; the sample outlet is connected to a product collecting tank, a flow channel and a plurality of detection chambers are arranged at the lower part of the product collecting tank, a control valve is arranged at the connection between the product collecting tank and the flow channel; an exosome outlet is arranged at the bottom of the detection chamber.

[0008] Preferably, a screen-printed electrode is provided in the detection chamber, and the screen-printed electrode includes a working electrode, a counter electrode and a reference electrode. The working electrode is obtained by growing gold nanoparticles and exosome probes on the surface of a glassy carbon electrode, the carbon ring electrode is the counter electrode, and the reference electrode is an Ag / AgCl electrode.

[0009] Compared with the prior art, the advantages and positive effects of the present invention are: The present invention proposes a new method for selectively enriching exosomes using bubbles as carriers. Zero-damage, ultra-fast and economical sorting and enrichment of tumor exosomes are achieved, avoiding the destruction of exosomes by strong physical fields. The bubble carrier can be continuously supplied through the inflation system, avoiding the complex and time-consuming synthesis process of nanomaterial carriers, and at the same time helping to improve the capture and recovery rate of exosomes. After the sorting is completed, the bubble carrier ruptures and disappears, without any contamination of the exosome product, solving the problem of magnetic bead carriers affecting downstream analysis, and realizing rapid and automated detection of "sample in-result out". BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is a schematic diagram of the device of the present invention.

[0011] Figure 2This is the liquid chromatogram of E-Am synthesized in the present invention.

[0012] Figure 3 This is the mass spectrum of E-Am synthesized by the present invention.

[0013] The reference numerals of the figures are: 1 bubble generator, 2 gas inlet, 3 sample storage area, 4 foam purification area, 5 sample outlet, 6 sample addition port, 7 product collection tank, 8 flow channel, 9 detection chamber, 10 control valve, 11 exosome outlet, 12 EP tube tank. DETAILED DESCRIPTION

[0014] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0015] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0016] Example 1 like Figure 1 As shown, this embodiment provides an exosome enrichment device, the device as a whole is a frame structure. Figure 1Taking the orientation shown as an example, the left side of the device is the separation and enrichment component of the exosomes, and the right side is the collection and detection component of the exosomes. Among them, a bubble generator 1 is provided at the bottom of the left side of the device. The aperture of the bubble generator 1 selected in this embodiment is 500 μm. A gas inlet 2 is provided on the side of the bubble generator 1. The gas inlet 2 is connected to the air pump through a pipeline. The upper part of the bubble generator 1 is a vertical hollow columnar sample storage area 3 and a foam purification area 4. The sample storage area 1 and the foam purification area 4 are connected to provide sufficient space for the generation and purification of foam. The upper end of the foam purification area 4 is provided with an exosome injection port 6. In order to prevent the foam from overflowing directly from the injection port 6, the injection port 6 is provided with a sealing cover. After the exosome sample is injected, the sealing cover is closed. Below the injection port 6, a sample outlet 5 is provided on the right side of the upper part of the foam purification area 4. The purified foam is discharged from the outlet. The sample outlet 5 structure can be set as a thin sheet with the upper end hinged on the upper wall of the device, and can only be opened toward the product collection tank on the right side. When the gas is not introduced, the sample outlet 5 is closed, and the exosome sample falls into the sample storage area 3 under the action of gravity; when the gas is introduced, the sample outlet 5 is opened under the impact of the airflow, and the foam enters the product collection tank 7 with the airflow. The product collection tank 7 is wide at the top and narrow at the bottom. After the foam continuously enters, it gradually gathers downward under the action of gravity. The bottom of the product collection tank 7 is connected to the exosome flow channel 8 through a pipeline adapter, and control valves 10 are set at the entrance of the exosome flow channel 8, the upstream and downstream channels of the detection chamber, and the flow channel 8 is connected to a number of detection chambers 9. Figure 1 There are four detection chambers 9 shown, and other numbers or distributions of detection chambers 9 can also be designed as needed. Electrodes for matching detection are arranged in the detection chamber 9. This embodiment adopts a three-electrode system, including a working electrode, a carbon ring electrode as a counter electrode, an Ag / AgCl reference electrode, and a glassy carbon electrode with gold nanoparticles and corresponding probes grown on the surface as a working electrode. The outlet of the detection chamber 9 is connected to the exosome outlet 11 through a flow channel 8. An EP pipe groove 12 is arranged directly below the exosome outlet 11 to receive the exosomes discharged after detection. When the collected exosomes are used for other downstream analyses, the detection electrode can be removed, and no reaction occurs after flowing through the detection chamber, and the sample outlet is directly further transported to the sample outlet for collection and standby.

[0017] The detection chamber can be designed and adjusted according to the actual detection task. This embodiment provides a method for preparing a working electrode: immerse a glassy carbon electrode in a mixed solution containing 1 g / L HAuCl4 and 100 mg / L KNO3, use a carbon ring electrode as a counter electrode, and Ag / AgCl as a reference electrode, and bubble pure N2 for 30 minutes to remove oxygen from the mixed solution. Then set the scanning speed to 50 mV s -1Under the condition of potential range of -0.1 V to 0.2 V, gold particles were grown on the surface of glassy carbon electrode for 60 s, and then the working electrode was taken out and blown dry with flowing N2 at room temperature for 15 min to remove moisture from the outer surface of the modified layer.

[0018] The exosome probe (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was diluted to 1 μM using TE buffer (containing 10 mmol / L Tris-HCl and 1 mmol / L EDTA). Then, 1 μL of 1 mM tris(2-chloroethyl) phosphate (TCEP) was mixed with 99 μL of 1 μM probe solution and reacted at room temperature for 40 min. 5 μL of the reacted probe solution was added dropwise to the working electrode surface and reacted at room temperature for 60 min. Then, the electrode was slowly rinsed with PBS buffer with a phosphate concentration of 0.1 M to remove the free probe, completing the modification of the probe on the electrode.

[0019] Example 2 This embodiment provides a specific process for exosome detection. First, 200 μL of lung cancer exosome serum (samples are from Qilu Hospital of Shandong University) is mixed with 10 mL of PBS buffer with a phosphate concentration of 0.1 M and 2 μL of E-Am molecules with a concentration of 5 μg / mL. E-Am targets the tumor exosome membrane surface protein CD63 and then binds to the exosome surface. The mixture is then added to the exosome storage area from the exosome sample injection port. After the air pump compresses the air, it enters the bubble generator through the gas input port of the bubble generator. The contact surface between the bubble generator and the exosome sample is a porous array structure with a pore size of 500 μm. The gas flows through each channel to generate bubbles on the contact surface. During the rising process of bubbles, they collide with tumor exosomes in the serum. E-Am molecules assist tumor exosomes to bind to the surface of bubbles and enhance the stability of bubbles. Other vesicles cannot form effective attachment with bubbles. After bubbles float out of serum, foam groups are formed above the serum surface. With the continuous input of bubbles, the height of the foam increases. In the foam product purification area, some unstable bubbles burst, and non-exosome vesicles mixed in the bubble interface fall off, achieving the purpose of further purifying the exosome product. When the exosome product reaches the exosome product outlet, it enters the exosome product collection tank, where the bubbles gradually burst to form a liquid containing tumor exosomes. The liquid is transported to each detection chamber through the sample flow channel. A screen-printed electrode is installed in each detection chamber, and a marker probe targeting lung cancer exosomes is modified on the surface of the working electrode. When the exosomes containing the marker are captured on the electrode surface, it will cause a change in the electrochemical signal, thereby obtaining the detection signal of tumor exosomes. The exosome liquid after detection is transported to the EP tube through the sample outlet.

[0020] The E-Am molecule used in this embodiment is synthesized by the following steps. In the following steps, unless otherwise specified, the material ratio is the mass ratio, and the percentage content of the material is the mass fraction: (1) Resin swelling Weigh 0.8 g of 2-chlorotrityl chloride resin (Aladdin) with a substitution degree of 0.3 mmol / g, put the resin into a reaction tube, add 10 mL of dichloromethane with a concentration of 15 ml / g, and shake for 30 minutes.

[0021] (2) Connect the first amino acid The solvent was filtered off through a sand core, and leucine (Leu) contained in 9-fluorenylmethoxycarbonyl (Fmoc) was added in an excess of 3 times the molar amount of the resin, and then 10 times the molar amount of N,N-diisopropylethylamine (DIEA) was added, and finally 3 mL of N,N-dimethylformamide (DMF) was added to dissolve, and shaken for 1 hour. Wash with N,N-dimethylformamide and dichloromethane alternately for 6 times.

[0022] (3) Deprotection Add 15 ml of 20% piperidine DMF solution, react for 5 min, pour off the upper liquid, add another 15 ml of 20% piperidine DMF solution, and react for 15 min.

[0023] (4) Detection Drain the piperidine solution, take a dozen resins, wash them three times with ethanol, add 3 drops of Kaiser Test detection solution (A solution: 20% anhydrous ethanol + 80% phenol, B solution: pyridine, C solution: 5g ninhydrin + 100ml anhydrous ethanol, there is no order for adding during detection, one drop of each), heat at 105-110℃ for 5min, and turn dark blue for a positive reaction.

[0024] (5) Wash The sample was washed twice with a DMF solution (10 ml / g), twice with a methanol solution (10 ml / g), and twice with a DMF solution (10 ml / g).

[0025] (6) Condensation A three-fold excess of Fmoc-protected arginine and a three-fold excess of O-benzotriazole-tetramethyluronium hexafluorophosphate were dissolved in as little DMF as possible and added to the reaction tube. A ten-fold excess of DIEA was immediately added and the reaction was continued for 40 minutes.

[0026] (7) Wash The sample was washed once with a DMF solution (10 ml / g), twice with a methanol solution (10 ml / g), and twice with a DMF solution (10 ml / g).

[0027] (8) Repeat steps (2) to (7) to connect the amino acids in the peptide chain sequence of the E-Am molecule from right to left.

[0028] (9) Detection Wash twice with DMF solution (10 ml / g), twice with DCM solution (10 ml / g), twice with DMF solution (10 ml / g), and drain for 10 min. Ninhydrin test was negative.

[0029] (10) Wash The mixture was washed three times with methanol solution (10 ml / g).

[0030] (11) Cleavage of peptides from resin Prepare 10g of cutting fluid: 94% trifluoroacetic anhydride; 2.5% water; 2.5% ethylenediaminetetraacetic acid; 1% triisopropylsilane.

[0031] The prepared resin was placed in a flask, and a cutting solution was added so that the ratio of the resin in the cutting solution was 10 ml / g. The mixture was shaken at a constant temperature of 40° C. for 120 min to obtain a cleavage solution.

[0032] (12) Blow dry and wash The lysate was blown dry with nitrogen as much as possible (for more than 1 h), 10 ml of ether was added for extraction, and then washed with ether six times, and then allowed to stand at room temperature for more than 2 h to obtain a crude peptide sequence.

[0033] (13) Purification of peptides by HPLC Specific steps: Take 200 mg of crude peptide and put it into a container, add 5 ml of 50% acetonitrile aqueous solution, and ultrasonicate for 2 minutes.

[0034] Filter with a 0.45 μm filter membrane.

[0035] Analysis: Take 3 μl and analyze the crude product by analytical HPLC. The mobile phase is water and acetonitrile, the time is 30 minutes, and the gradient elution is performed. The HPLC is equilibrated with a starting gradient for 5 minutes before injection. The starting gradient is 95% water, 5% acetonitrile, and the ending ratio is 5% water, 95% acetonitrile.

[0036] Preparation: Prepare the dissolved sample for injection. Prepare HPLC for 10 minutes, start gradient: 95% water, 5% acetonitrile, end gradient: 25% water, 75% acetonitrile, gradient time: 40 minutes. Collect the sample from the detector.

[0037] Identification: The collected samples were sampled for purity and MS identification. The results were as follows: Figure 2 and Figure 3 As shown, from Figure 2 and Figure 3It can be seen that the E-Am molecule was successfully synthesized with a purity of 98.6%.

[0038] (14) Finally, the purified solution is freeze-dried to obtain the finished product.

[0039] (15) The powdered E-Am molecules are sealed and stored at -20 degrees.

[0040] The traditional process for purifying exosomes is ultracentrifugation, but ultracentrifugation has low separation efficiency, cumbersome operation, high requirements for equipment, and strong centrifugal force damages the structure of exosomes, affecting the activity of exosomes. Due to the small size of exosomes (30-150nm) and low buoyancy density, it is difficult to separate from a complex multi-vesicle system. The present invention uses bubbles as carriers, proposes a new technology for efficiently enriching tumor exosomes, and designs E-Am functional molecules that bind to exosomes based on the gas-liquid interface microenvironment in the solution (without adding this molecule, bubbles cannot effectively transport exosomes), and constructs an integrated system for exosome transport and exosome detection. Exosomes are attached to the surface of bubbles and are automatically transported to the exosome product collection area under the action of buoyancy. The system can obtain exosome samples enriched 50-100 times within five minutes. The obtained exosome products can be used for detection of the device system of the present invention, and can also be flexibly used for other analyses (this is currently based on magnetic nanoparticle immunoaffinity and microfluidics to obtain exosomes. The technology cannot be achieved, and they are difficult to elute). The device does not require complicated external equipment configuration during operation, and there is no strong electric field force, centrifugal force and acoustic force to damage exosomes, which provides a new idea for the efficient sorting and enrichment of micron and nanometer-scale biological particles. The process proposed by the present invention, which uses bubbles as carriers and sorts biological microparticles based on the characteristics of the gas-liquid two-phase interface, is a new sorting technology in the biomedical field.

[0041] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Application of E-Am molecules in exosome enrichment and / or detection, characterized in that: The molecular formula of the E-Am class molecule is as follows: R1-R2-R3-COOH; wherein R1 is a C15 straight chain alkyl group; R2 is ; R3 is the following peptide chain: -Cys-Arg-His-Ser-Gln-Met-Thr-Val-Thr-Ser-Arg-Leu-, Among them, Cys is cysteine, Arg is arginine, His is histidine, Ser is serine, Gln is glutamine, Met is methionine, Thr is threonine, Val is valine, and Leu is leucine.

2. The application according to claim 1, characterized in that: The application method is: exosome serum is mixed with PBS and E-Am molecules to obtain a mixed solution, high-pressure bubbles are introduced from the bottom of the mixed solution and then float to the surface to form foam, high-pressure bubbles are continuously introduced, the foam rises, and the stable foam group formed after rising to a certain height is collected to complete the enrichment of exosomes. The enriched exosomes are collected and stored for later use or detection.

3. The application according to claim 1, characterized in that: The volume ratio of exosome serum: PBS: E-Am molecules is (50-200): (1000-50000):

1.

4. A device based on the application according to any one of claims 1 to 3, characterized in that: It includes a bubble generator, a gas inlet is arranged on the side of the bubble generator, a cylindrical sample storage area and a foam purification area are arranged in sequence above the bubble generator, a sample outlet is arranged on the side of the top of the foam purification area, and a sample addition port is arranged on the upper part of the foam purification area; the sample outlet is connected to a product collection tank, a flow channel and a plurality of detection chambers are arranged at the lower part of the product collection tank, and a control valve is arranged at the connection between the product collection tank and the flow channel; an exosome outlet is arranged at the bottom of the detection chamber.

5. The device according to claim 4, characterized in that: The detection chamber is provided with a screen-printed electrode, which includes a working electrode, a counter electrode and a reference electrode. The working electrode is obtained by growing gold nanoparticles and exosome probes on the surface of a glassy carbon electrode, the counter electrode is a carbon ring electrode, and the reference electrode is an Ag / AgCl electrode.

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